Tyrosine kinase inhibitors as antiproliferative agents against an estrogen-dependent breast cancer cell line in vitro.

摘要

BACKGROUND AND OBJECTIVES: Receptor tyrosine kinase (RTK) activation is critical for growth factor-mediated cell proliferation. Blockade of RTK activation inhibits growth factor-induced cell proliferation. A panel of RTK inhibitors (tyrphostins) have been tested and compared for their antiproliferative effects on the hormone-dependent human breast cancer cell line, MCF-7, in vitro. METHODS: MCF-7 cells (10(4)/well) were seeded into 96 well plates and maintained in DMEM with 1% bovine serum albumin (BSA), 200-pg/mL estrogen, or 10% fetal bovine serum. After a defined time interval, the cells were exposed to RTK inhibitors and a non-RTK-inhibitory analog of tyrphostins (0 to 400 microM). After 3 days, the number of viable cells in each well was estimated by an MTT assay and the results expressed as percent of controls. Using a representative tyrphostin, A47, the validity of MTT assay as a measure of cell proliferation was tested by a colony formation assay and by immunostaining with Ki-67 antibodies. RESULTS: MCF-7 cells maintained in DMEM containing 1% BSA without E2 or serum showed a minimal increase in cell number. Supplementation with E2 stimulated cell proliferation in a dose-dependent manner. This E2-mediated growth stimulation was completely inhibited (cytostatic effects) by the epidermal growth factor receptor (EGFR)-selective tyrphostins A47, B48, RG13022, and B50. These same tyrphostins also decreased the cell numbers to below control numbers in cultures maintained in 1% BSA or in serum containing medium (cytostatic/cytotoxic effects). B44 (EGFR-selective tyrphostin), AG1295 (platelet-derived growth factor receptor [PDGFR]-selective tyrphostin), and A1 had no inhibitory effects on cells with or without E2 treatments. However, A1 inhibited cell growth under serum supplementation. Genistein, a phytoestrogen, stimulated the autonomous, E2-induced as well as serum-induced growth of MCF-7 cells. Cell proliferation results derived from the MTT assay were corroborated by both the colony formation assay as well as the Ki-67 assay. CONCLUSIONS: Of the agents tested, only EGFR-selective tyrphostins blocked E2-stimulated tumor cell proliferation, as opposed to the PDGFR-selective tyrphostin, RTK noninhibitory agent, or the phytoestrogen, genistein, which did not exert such an effect. These findings suggest that epidermal growth factor (EGF) is an important mediator of E2-induced proliferation of MCF-7 cells. Thus, tyrphostins may be selectively used to prevent the growth of hormone-dependent breast cancers, particularly regrowth of residual tumor in postmenopausal breast cancer survivors receiving estrogen replacement therapy.